Published April 1, 1970
CONTROLLED PRODUCTION OF
PROLIFERATING SOMATIC CELL HYBRIDS
ROBERT J . KLEBE, TCHAW-REN CHEN,
and
FRANK H . RUDDLE
From the Department of Biology, Kline Biology Tower, Yale University,
New Haven, Connecticut 06520
ABSTRACT
INTRODUCTION
Somatic cell hybridization of mammalian cells involves the fusion of parental cells to produce a
single proliferating hybrid cell with the genetic
complement of both parental cells (15) . Okada
and co-workers (17-19) have demonstrated that
live Sendai virus can be used to fuse suspensions of
mononucleated tissue culture cells to yield nonproliferating single cells which contain large numbers of nuclei . The feasibility of using UV-inactivated Sendai virus in promoting the production
of growing somatic cell hybrids was first described
by Yerganian and Nell (25) . Heretofore, somatic
cell hybridization relied on chemical selection for
infrequent spontaneous fusion events (15) . The
techniques to be described permit the controlled
quantitative production of large numbers of growing hybrid clones with the use of convenient numbers of cells and an uncomplicated methodology .
MATERIALS AND METHODS
Preparation of Inactivated Sendai Virus
Sendai virus (Para-influenza I) was grown and
harvested according to Okada, Murayama, and
74
Yamada (19) . In brief the method is as follows : 0 .2
ml of a 10-s dilution of infected allantoic fluid
[1000 hemagglutinating units of virus (HAU)/ml]
was inoculated into the allantoic cavity of 9-day
embryonated eggs . After 72 hr, the eggs were placed
in the cold room at 7 °C for 12 hr to induce clotting
in order to prevent bleeding during the harvesting
procedure . Yields of 1000 HAU/ml or more were
routinely obtained as determined by the Salk pattern
method (3), employing 0.5 ml of 0 .5 1/0 chicken red
blood cells with phosphate-buffered saline (PBS) as
diluent and 0 .5 ml of two-fold dilutions of infected allantoic fluid . Titrations were carried out in 96-U-WS
Disposo-Trays (Linbro Chemical Co ., New Haven,
Conn .) . The infected allantoic fluid was clarified at
2000 g for 20 min and the virus was pelleted at
16,000 g for 1 hr . The pellet was resuspended in onetenth the starting volume with PBS + 1 /0 bovine serum albumin (Sigma Chemical Co ., St . Louis, Mo.) .
The addition of bovine serum albumin at this step prevents a two-fold decrease in virus titer during the inactivation step . This material was clarified again and immediately inactivated with ß-propriolactone (ßprone, Fellows Testagar, Detroit, Mich.) as described
by Neff and Enders (16) . ß-prone was diluted to 10%
with triple glass-distilled water and then the 10%
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The techniques described permit the controlled production of large numbers of proliferating somatic cell hybrids in a relatively short period of time . Sendai virus is used to promote cell hybridization . ß-propriolactone is employed as the inactivating agent of Sendai
virus since it produces complete loss of viral infectivity while preserving viral fusion capacity . Cells are fused in monolayer, instead of in suspension, since fixing cells in two dimensions permits one to control cell contacts during the fusion event through the expedient of
varying multiplicities of the parental cells and the total cell density . Under the conditions
described, a several hundred fold increase in the number of hybrid clones obtained is seen
as compared to the controls.
Published April 1, 1970
aqueous solution was diluted to 2% with a saline
bicarbonate solution (1 .68 gm of NaHCO5 + 0 .5
ml of 0.4% phenol red + 100 ml of isotonic NaCl) .
A virus suspension containing 10,000 HAU/m1 was
brought to a final concentration of 0 .05% iß-prone
with the above 2% /(3-prone solution and incubated
with shaking at 4 °C for 10 min, at 37 °C for 2 hr to
inactivate the virus, and at 4 ° C overnight to insure
the complete hydrolysis of the remaining 0-prone .
The inactivated virus was diluted to 2000 HAU/ml
with culture medium (Dulbecco-Vogt (24) modified
Eagle's medium + 10% agamma calf serum + 100
units/ml penicillin + 100 jug/nil streptomycin
[GIBCO, Grand Island, N. Y.]) and frozen at
-70 °C in ampoules .
Adaptation of Cells to Tissue Culture and
Isolation of Mutants
Biochemical Selection of Somatic
Cell Hybrids
The biochemical selection technique of Littlefield
(15) was employed . The selection medium consists of
Dulbecco-Vogt medium + 10% agamma calf
serum + 100 units/ml penicillin + 100 µg/ml
streptomycin, supplemented with 1 .6 X 10-5 M
thymidine, 10-4 M hypoxanthine, and 4 X 1007 M
aminopterin (15) . In brief, the basis of the selection
procedure is the following . Aminopterin inhibits
dihydrofolate reductase which converts dihydrofolate
to tetrahydrofolate (12) . In the absence of the cofactor tetrahydrofolate, the de novo synthesis of purines
is blocked (12) and, in addition, the methylation of
dUMP to dTMP by thymidylate synthetase (EC
2 .1 .1 .b) (12) and the conversion of serine to glycine
by serine transhydromethylase (EC 2 .1 .2 .1 .) (12) are
inhibited . Cells treated with aminopterin proliferate
if exogenous hypoxanthine, thymidine, and glycine
(supplied in Dulbecco-Vogt medium [24]) are added
(10) . The conversion of hypoxanthine into purines is
catalyzed by hypoxanthine-guanine phosphoribosyl
transferase (HG-PRT) (EC 2.4 .2 .8) . Exogenous
hypoxanthine is not utilized by 8-azaguanine-resistant mutants (22), since HG-PRT is reduced in
activity or lacking (22) . Cells resistant to 5'-bromodeoxyuridine (BUdR) do not utilize exogenous thymidine (8) due to the lack of the enzyme thymidine
kinase (EC 2 .7 .1 .21) (8) . Thus, in Dulbecco-Vogt
HAT medium, the parental 8-azaguanine-resistant
and BUdR-resistant cells die while somatic cell
hybrids between these mutants proliferate (15) due
to intergenic complementation .
Monolayer Fusion Procedure
In the experiments to be described a nonreverting
8-azaguanine-resistant renal adenocarcinoma, RAG,
derived from the BALB/cd strain of mice (described
above), and a nonreverting BUdR-resistant L-cell
LM(TK ), (13) derived from a C3H mouse were
studied ; however, similar results have been obtained
with several other pairs of cell lines . The fusion
indices (F.I .) (18) of the RAG and LM(TK) cell
lines are similar, being 0 .6 and 0 .4, respectively, at
1000 HAU of inactivated virus under Okada's conditions (18) . In all cases, stationary phase populations were used as starting material since it has been
reported that cells in interphase may undergo chromosome pulverization as a result of fusion with a cell
in mitosis (23) . At the time of the addition of virus,
cells were in mid-lag phase as deduced from their
growth curves.
KLEBE, CHEN, AND RUDDLE
Production
of Somatic
Cell Hybrids
75
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We would like to acknowledge the gracious
assistance of Dr. Albert Claude in providing us with
a transplantable renal adenocarcinoma derived from
the BALB/cd strain of mice in which renal adenocarcinoma occurs at a high incidence in adults (9) .
The tumor cells were adapted to in vitro culture by
alternate animal-to-tissue culture passage (2) . Tumor
tissue was fragmented mechanically into small
clumps and single cells, and was planted in F-12
medium + 10% agamma calf serum + 100 units/ml
penicillin + 100 µg/ml streptomycin (GIBCO) .
After 1 wk in culture, the surviving cells were inoculated into the appropriate host and the process was
repeated again. After two animal-to-tissue culture
passages, a population of proliferating renal adenocarcinoma cells was obtained which, however, was
contaminated by normal fibroblasts of stromal
origin . The renal adenocarcinoma cells were freed
of the contaminating fibroblasts by growing the
mixed population in suspension in untreated plastic
bacteriological petri plates . Under these conditions,
the contact-inhibited normal cells perish while the
non-contact-inhibited renal adenocarcinoma cells
proliferate . Since the renal adenocarcinoma forms
diffuse colonies when seeded at low density in monolayer, the cell population was serially cloned three
times in 0 .125% semisolid agarose (5) . The cloned
population of renal adenocarcinoma cells is termed
Renal. All subsequent experiments were performed
with Dulbecco-Vogt medium.
A nonreverting 8-azaguanine resistant mutant was
obtained by exposing 107 cells to 5 µg/ml of 8-azaguanine (AG) (Mann Research Laboratories, New
York, N .Y.) and then by exposing the survivors to
20 µg/ml of the drug . The resistant mass population
was then cloned and each clone derived was tested
for revertants in the selection medium HAT (15) .
One clone, termed RAG, was identified which has
proven to be free of revertants despite prolonged
growth in culture medium lacking the drug . This
cell line has been submitted to the American Type
Culture Collection.
Published April 1, 1970
Following trypsinization and resuspension in fresh
medium, a predominantly single cell suspension of
the two cell lines was mixed in culture tubes at
multiplicities and densities appropriate to the experiment prior to being planted in Falcon T-30
flasks (Falcon Plastics, Los Angeles, Calif.) (surface
area = 25 cm) . The culture flasks were incubated
for 16 hr at 37°C to allow the cells to attach and
spread with a minimum of cell division . Once the
cells had spread, the medium was removed and the
flask was placed at 7 °C for 10 min. 1 ml of chilled
virus suspension was then added and the cells were
left at 7 ° C for another 20 min to permit viral adsorption . At the end of the adsorption period, 4 ml
of warm culture medium was added, and the cultures were immediately returned to the 37 ° C incubator for 2 hr . At the end of this time, the medium
was removed and the cell sheet was washed twice
with culture medium before the selection medium,
HAT, was added. In this medium, only fusion products between parental cells can grow, whereas the
parental cells die . After 10 days in HAT medium,
the culture flasks were washed with isotonic saline,
air dried, and stained with Wright stain for colony
counting .
Biochemical and Cytogenetic Methods
Employed in Characterizing the Somatic
Cell Hybrids Produced with Inactivated
Sendai Virus
The starch gel assay for glucose-6-phosphate isomerase (EC 5.3 .1 .9) has been described by DeLorenzo and Ruddle (7) .
Chromosome preparations were made according
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Phase-contrast photomicrographs of the parental and hybrid cell lines . Rag has an ameboid
to epithelioid morphology with prominent cytoplasmic processes ; LM(TB) displays a characteristic
cobble-stone growth pattern ; and RELM appears to possess morphological properties which are the
intermediate of the parental cell lines . Note the large number of prominent nucleoli in the hybrid cells.
FIGURE 1
76
THE JOURNAL OF CELL BIOLOGY • VOLUME 45, 1970
Published April 1, 1970
to the method described by Chen (4) . Semiconfluent
cultures were treated with a final concentration of 1
,ug/ml of colcemid (CIBA Products Company,
Fair Lawn, N .J .) for 4 to 5 hr, collected with hypotonic trypsin-versene solution (2 g/liter NaCI, 0.2
g/liter KCI, 0 .2 g/liter KH 2 PO4 , 1 .15 g/liter
Na2 HPO4 , 1 g/liter glucose, 0.10 g/liter trypsin
1 :250 (Difco Laboratories, Detroit, Mich.), and 1 .0
g/liter disodium versene), swollen in hypotonie KCI
(0.075 M) for 6 min, pelleted, and fixed twice for 15
min with 1 :3 acetic acid-methanol . Slides were prepared by air drying and were stained with 1 .5%
aceto-orcein .
2 LM(TK-) karyogram. Arrows indicate
three marker chromosomes (L-1, L-2, and L-3) which
are readily distinguishable in the hybrid metaphase
plate .
FIGURE
RESULTS
Hybrid Characteristics
were present in each independently derived clone
(Figs . 2-4) and, secondly, the expression of the
3 RAG karyogram. Arrows indicate two
marker chromosomes (R-1 and R-2) .
FIGURE
parental and heteropolymeric forms of the glucose6-phosphate isomerase isozymes serves to indicate
the presence in the hybrid cells of allelic genes
derived from the BALB/cd- and C3H-derived
parental cell lines (Fig. 5) . The hybrids of the
renal adenocarcinoma and the LM(TK -) cell
lines have been designated RELM followed by a
number indicating their clonai origin .
Metaphase plate of a hybrid cell [LM(TK- ) X RAG .] Arrows
demonstrate marker chromosomes derived from both parental cell lines .
FIGURE 4
KLEBE, CHEN, AND RUDDLE
Production of Somatic Cell Hybrids
77
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After 3-4 days in HAT medium, the parental
cells had rounded up and begun to detach from
the substratum ; and within 7 days, colonies of cells
appeared that were larger than either of the parents and morphologically dissimilar (Fig . 1) .
Several of these colonies were cloned and characterized as hybrids on two grounds . First, marker
chromosomes derived from each of the parents
Published April 1, 1970
Quantitation of Cell Hybridization
The parental cell lines were mixed in a 1 : 1
multiplicity and then planted at 5 X 10 5, 10 6, and
2 X 10 6 total cells/25 cmz surface area culture flask
and treated with inactivated virus as described
previously . The yields of proliferating hybrid
clones is dependent on the titer of virus employed
and increases as the cell density increases (Fig .
6) . The number of hybrid clones produced increases as the ratio of virus particles to cells
increases up to a plateau at 2000 HAU, as shown
in Fig. 7 . Above 2000 HAU, a slight decline in the
number of virus-promoted hybrid clones is encountered ; and this effect is most pronounced at
the lowest cell density. The decrease in hybrids at
low cell density and high virus titer may be due
to the inability of cells to repair more than a
certain number of virus-induced cell surface lesions
in a given period of time . It should be noted that
78
the ability of Sendai virus to fuse cells falls off
above 1000-5000 HAU (18) . When the parental
cell lines are mixed in different multiplicities, the
optimal yield of hybrid clones occurs at or near a
1 :1 ratio of the parents (Fig . 8) .
Quantitatively, no deterioration of the ability of
$-prone inactivated virus to produce growing hybrids occurs over a period of 5 months when the
virus is stored ampouled at -70°C (Table I) .
Ampoules of virus are frozen rapidly to -70 ° C
and thawed rapidly to 37 ° C .
DISCUSSION
In order to obtain large numbers of growing somatic cell hybrid clones, the viral fusion method of
Okada and co-workers (18), the viral inactivation
procedure of Neff and Enders (16), and the monolayer fusion stratagem of Kohn (14) have been
employed for the reasons cited below .
THE JOURNAL OF CELL BIOLOGY - VOLUME 45, 1970
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Glucose phosphate isomerase isozyme patterns . Channel 1, BALB/cJ kidney homogenate :
Channel .G, BALB/c derived renal adenocarcinoma cell culture ; Channels 3-7, hybrids ; Channel 8, C3H
derived cell line ; Channel 9, C3H kidney homogenate . Note that the cell cultures retain isozyme patterns
distinctive of the mouse strain of origin and also note that the independently derived hybrid clones
express the isozyme patterns of both cell lines and a band migrating at an intermediate rate between the
parental bands, indicating the formation of a heteropolymeric molecule with subunits coded by both
parental genomes .
FIGURE 5
Published April 1, 1970
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6 Hybrid clones produced at varying virus titer and cell densities . Note particularly the heterogeneity in clonal sizes .
FiGUBE
First, Harris et al . (11) report that UV inactivation reduces viral infectivity by a factor of 10 6 ,
while it has been our experience and that of Neff
and Enders (16) that ß-prone completely inactivates Sendai virus . Since 1 HAU of Sendai virus
represents 2 .4 X 10 1 virus particles (17) and since
the particle-to-infectivity ratio for Sendai virus is
about 10 :1 (17), one would be introducing approximately 2 .4 X 102 live virus particles in a
hybridization experiment employing only 100
HAU of UV-inactivated virus . Infection of cells
at a low multiplicity of infection can lead to the
permanent residence of para-influenza viruses in
their host in a carrier state without loss of cell
viability or noticeable cytopathic effects (21), but
with the chance of chromosomal abnormalities
(23) . Chemical inactivation of Sendai virus has
the double advantage of technical simplicity and
of producing complete viral inactivation . ß-propriolactone is an alkylating agent which reacts with
the guanosine moiety in the viral RNA when the
reaction is carried out in a slightly basic environment (20) . Since the para-influenza group of
myxoviruses does not exhibit multiplicity reactivation (I), hybridization experiments, which by
necessity must employ a high multiplicity of virus
KLEBE, CHEN, AND RUDDLE
Production of Somatic Cell Hybrids
79
Published April 1, 1970
10
3
N
= 10
2
o
U
0
K
•
r
•
Io
0
10
''
""10
02
VIRUS TITER
""10 3
(HAU)
4
Relationship between virus titer employed
and yield of hybrid clones . (0), 2 X 10 6 total cells ;
(0), 10 6 total cells ; (A) 5 X 106 total cells .
FIGURE 7
40
which are nearest neighbors . The nearest neighbor
of a given cell can be predetermined statistically
by varying the multiplicity of one parent cell line
versus the other parent and can be manipulated
still further by the cell density employed . At low
cell density and at a multiplicity of one cell A to
one cell B, the nearest neighbor of cell A would
most probably be cell B.
In a similar study (6), cells were fused in suspension . Under these conditions, the extent of
hybridization did not appreciably increase with
increasing virus concentration . Possibly the formation of nonviable multiple fusion products during
fusion in suspension has decreased the extent of
hybridization with increased virus titer .
In employing the monolayer fusion method, the
following recommendations may be useful . If the
fusion index of the parental lines is similar and low
at 1000 HAU, the parents should be mixed in a
1 :1 multiplicity, planted at a high cell density,
36
Effect of Storage Time at -70°C on Ability of Inactivated
Sendai Virus to Promote Cell Hybridization
28
u)
Z
-J 24
U
n
20
B
i
16
500
250
100
0
12
Freshly
inactivated
virus
Inactivated
virus stored
for 5 months
190
175
79
5
182
161
78
3
HAU
HAU
HAU
HAU
Parental cell lines were mixed at a 1 :1 multiplicity
and planted at a density of 2 X 10 6 total cells/25 cm-'
surface area flask .
8
0 HAU
I
MULTIPLICITY
10
Relationship between input multiplicity of
parental cells and yield of hybrid clones at constant
cell density and virus titer. All points represent a total
cell density of 106 cells . (D), 500 HAU employed in
fusion ; (0), 100 HAU; (A), 0 HAU .
FIGURE 8
particles to cells, do not run the risk of yielding
live virus as a result of genetic crossing-over between virus genomes with lesions at different sites .
Secondly, hybridization of cells in monolayer
allows one several controls over the extent of
fusion and kinds of progeny derived which are not
available when cells are fused in suspension . Fixing
cells in two dimensions restricts fusion to cells
80
10
-
10
-4
( Ro9 /LM (TK-))
W
0
9
B~Q
0
0
°-
Z
N
N
0
K
Y
A
A
10-5
A
-61,
""
10
10
10
2
VIRUS TITER
10
3
10
4
(HAU)
Relationship between virus titer and hybridization index . (D), 2 X 10 6 total cells ; (0), 106
total cells ; (A), 5 X 106 total cello .
FIGURE 9
THE JOURNAL OF CELL BIOLOGY - VOLUME 45, 1970
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TABLE I
32
Published April 1, 1970
in a subpopulation consisting of 5 X 10 3 A-cells
and 5 X 10 3 B-cells, one particular fusion between
an A-cell and a B-cell gave rise to a colony of
hybrid cells . Since the relationship between the
fusion index and virus titer and between the hybridization index and virus titer are qualitatively
similar, the fusion index of a cell line should serve
as a useful gauge of its hybridization index .
The authors would like to express their appreciation
to Dr . Richard A . Goldsby for helpful advice in
handling Sendai virus . We thank Dr . Thomas B .
Shows and Miss Elizabeth Nichols for aid with the
starch gel assays, and also appreciate Miss Grace
Yun's skillful assistance in preparing the tissue culture reagents. In the preparation of the manuscript,
the excellent assistance of Mrs . Mae Reger is gratefully acknowledged.
This study was supported by U . S . Public Health
Service Predoctoral Fellowship 1-FI-GM-42,216-01
(awarded to Robert J . Klebe) and U . S. Public
Health Service Grant GM 09966-07 (awarded to Dr .
Frank H. Ruddle) .
Received for publication 30 July 1969, and in revised form
14 October 1969.
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DUBBS,
KLEBE, CHEN, AND RUDDLE
-Production of
Somatic Cell Hybrid8
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and fused with 1000 HAU or less . If both parents
have a high fusion index at 1000 HAU, the parents
should be mixed at a I : 1 multiplicity, seeded at a
low cell density, and fused with 1000 HAU or less .
If parent A has a much higher fusion index than
parent B, the multiplicity of A :B should be low,
and the cells should be planted at a low density
and treated with less than 1000 HAU to minimize
A X A X B fusions . After the period at 37 ° C,
virus-treated cells can be safely trypsinized and
replanted at low density in HAT medium in order
to obtain large numbers of independent clones .
Okada (18) has used the term fusion index
(F .I .) to describe the extent of cell fusion produced
by Sendai virus (F .I . = [number of cells prior to
fusion/number of cells after fusion] - 1) . The
term hybridization index (H.I . = the number of
hybrid clones produced by a given concentration
of virus/total cells employed at a 1 : 1 multiplicity)
is meaningful in describing the extent of hybridization and is nearly constant at a given dose of virus
at the cell densities employed here (Fig . 9) . A
hybridization index of 10 -4 would indicate that
Published April 1, 1970
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U.S.A . 55 :1066 .
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•
VOLUME 45, 1970